Parasiticidal milbemycin derivatives

ABSTRACT

Compounds of formula (I): ##STR1## wherein R 1  is hydrogen or methyl, R 2  is hydrogen or (E)-2-methyl 2-butenoyloxy, and R 3  is hydrogen or hydroxy, with the proviso that when R 3  is hydrogen, R 1  and R 2  are both hydrogen, and when R 2  is (E)-2-methyl 2-butenoyloxy, R 1  is methyl; the compound of formula (II): ##STR2## and the compound of formula (III): ##STR3## are obtainable by the fermentation of Streptomyces E225 NCIB 12310 or Streptomyces E225B NCIB 12509. The compounds have anthelmintic utility.

This application is a divisional of U.S. patent application Ser. No.08/457,809, filed Jun. 1, 1995, now U.S. Pat. No. 5,620,874, which inturn is a file-wrapper continuation of U.S. patent application Ser. No.08/303,889, filed Sep. 9, 1994, now abandoned, which in turn is afile-wrapper continuation of U.S. patent application Ser. No.08/108,790, filed Aug. 18, 1993, now abandoned, which in turn is afile-wrapper continuation of U.S. patent application Ser. No.07/496,112, filed Mar. 19, 1990, now abandoned, which in turn is afile-wrapper continuation of U.S. patent application Ser. No.07/076,274, filed Jul. 22, 1987, now abandoned.

The present invention relates to novel anthelmintically active materialsobtainable from a microorganism, to processes for their production, topharmaceutical formulations containing them, and to their use in humanor veterinary medicine.

A large number of microorganisms have been isolated, in particular fromsoil samples, and certain of those microorganisms have been found toproduce various metabolites, which can be isolated and some of whichhave useful biological activity. One group of such metabolites is themilbemycins, which have been prepared by the cultivation ofmicroorganisms of the genus Streptomyces and are described in inter aliaGB-A-1,390,336, J. Antibiotics 29(3), 76-14 to 76-16 and 29 (6), 76-35to 76-42, U.S. Pat. No. 4,144,352, and GB-A-2 056 986.

The α series of milbemycins include compounds of formula A: ##STR4##wherein R^(a) is methyl, ethyl or isopropyl. In U.S. Pat. No. 4,144,352it was disclosed that these and related compounds have anthelminticactivity.

Various milbemycin derivatives are disclosed in U.S. Pat. No. 4,093,629and U.S. Pat. No. 4,134,973.

EP-A-0 170 006 and GB-A-2 166 436 disclose six further compounds offormula B: ##STR5## wherein R^(b) is hydroxy or methoxy and R^(c) ismethyl, ethyl or isopropyl. These compounds were also prepared by thecultivation of Streptomyces microorganisms, and are stated to haveanthelmintic activity.

We have now discovered a new group of compounds obtainable by thecultivation of a Streptomyces microorganism. These compounds haveanthelmintic properties, and therefore are of use in the treatment ofhelminthiasis in humans and animals.

The present invention accordingly provides compounds of formula (I):##STR6## wherein R¹ is hydrogen or methyl, R² is hydrogen or(E)-2-methyl 2-butenoyloxy and R³ is hydrogen or hydroxy, with theproviso that when R₃ is hydrogen, R¹ and R² are both hydrogen, and whenR² is (E)-2-methyl 2-butenoyloxy, R¹ is methyl.

Compounds of formula (I) are set out in Table I below:

                  TABLE I                                                         ______________________________________                                        Compound  R.sup.1  R.sup.2          R.sup.3                                   ______________________________________                                        VM44857   H        H                H                                         VM44864   CH.sub.3 H                OH                                        VM44865   CH.sub.3                                                                                ##STR7##        OH                                        VM44866   H        H                OH                                        ______________________________________                                    

A further aspect of the invention provides VM 44867, which is thecompound of formula (II): ##STR8##

A still further aspect of the invention provides VM 44868, which is thecompound of formula (III): ##STR9##

Characterizing data for the compounds of the invention are set outhereinbelow in the Examples.

The present invention also provides a process for the production of acompound of the invention or a derivative thereof, which comprisescultivating a producing microorganism, and subsequently isolating thecompound or derivative thereof from the culture.

The present invention furthermore provides a process for the preparationof a compound of the invention or derivative thereof, which compriseschromatographically separating the compound or derivative thereof from asolution thereof in admixture with other substances into a fractioncomprising the compound or derivative thereof and other fractions.

The term `cultivation` (and derivatives of that term) as used hereinmeans the deliberate aerobic growth of an organism in the presence ofassimilable sources of carbon, nitrogen, sulphur and mineral salts. Suchaerobic growth may take place in a solid or semi-solid nutritive medium,or in a liquid medium in which the nutrients are dissolved or suspended.The cultivation may take place on an aerobic surface or by submergedculture. The nutritive medium may be composed of complex nutrients ormay be chemically defined.

It has been found that suitable microorganisms for use in thecultivation process according to the invention include bacterial strainsbelonging to the genus Streptomyces that are capable of elaboratingcompounds according to the invention. It has further been found thatexamples of such strains include Streptomyces E225, which has beenisolated from soil, and also mutants and natural variants thereof suchas Streptomyces E225B.

The term `mutant` as used herein includes any mutant strain which arisesspontaneously or through the effect of an external agent whether thatagent is applied deliberately or otherwise. Suitable methods ofproducing mutant strains include those outlined by H. I. Adler in`Techniques for the Development of Microorganisms` in "Radiation andRadioisotopes for Industrial Microorganisms", Proceedings of aSymposium, Vienna, 1973, page 241, International Atomic EngergyAuthority, and these include:

(i) Ionizing radiation (e.g. X-rays and γ-rays), u.v. light, u.v. lightplus a photosensitizing agent (e.g. 8-methoxypsoralen), nitrous acid,hydroxylamine, pyrimidine base analogues (e.g. 5-bromouracil),acridines, alkylating agents (e.g. mustard gas, ethyl-methanesulphonate), hydrogen peroxide, phenols, formaldehyde, nitrosoguanidine,heat, and

(ii) Genetic techniques, including, for example, recombination,transformation, transduction, lysogenisation, lysogenic conversion,protoplast fusion, and selective techniques for spontaneous mutants.

Streptomyces E225 and Streptomyces E225B are believed to comprise apreviously unreported strain in the genes Streptomyces and thereforeform part of the present invention, more particularly in biologicallypure form. They have been deposited in the National Collection ofIndustrial and Marine Bacteria, Aberdeen, Scotland, the deposits (NCIB12310 and 12509; filing dates 23rd Jul., 1986 and 20th Jul., 1987) beingmade under the terms of the Budapest Treaty on the InternationalRecognition of the Deposit of Micro-organisms for the Purposes of PatentProcedure.

The characteristics of Streptomyces E225 were as follows:

After being grown on starch casein agar medium for 7 to 14 days at 27°C., Streptomyces E225 had produced a yellow-brown vegetative mycelium inwhich the hyphae did not fragment into coccoid or bacillary elements,and a yellow or white aerial mycelium which turned grey as the cultureaged. The culture also produced a yellow soluble pigment and somecolonies were observed to exude yellow droplets. The sporophores werearranged singly or in pairs along straight or flexuous main aerialhyphae, with no evidence of true verticillate branching, and terminatedin spirals of 4 to 6 turns. Some sporophores presented a wartyappearance, whilst older cultures developed moist black areas wherespores had massed together.

Streptomyces E225 was non-sporulating on a yeast-malt agar medium.

The fermentation medium for cultivating the producing organism suitablycontains sources of assimilable carbon and assimilable nitrogen togetherwith inorganic salts. Suitable sources of nitrogen include yeastextract, soybean flour, meat extract, cottonseed flour, malt, distillersdried solubles, amino acids, protein hydrolysates and ammonium andnitrate nitrogen. Suitable carbon sources include glucose, lactose,maltose, starch and glycerol. Suitably the culture medium also includesalkali metal ions (for example, sodium), alkaline earth metal ions (forexample, calcium and magnesium), halogen ions (for example, chloride),and trace elements (for example, iron, zinc, copper, manganese andcobalt).

The cultivation may suitably be effected at a temperature of about 20°to 35° C., advantageously 27° to 28° C., and the culture may suitably beharvested after 2 to 35 days, advantageously about 5 to 20 days, afterthe initiation of fermentation in order to given an optimum yield of thedesired compound of the invention.

The desired compound or derivative thereof may then be isolated from theculture medium and worked up and purified using conventional techniques.

The desired product may be obtained from either the mycelial growth orfrom the culture filtrate. It may therefore be convenient for the firstisolation step to involve the separation of solid material from thefermentation broth by, for example, filtration or centrifugation, togive a clarified culture filtrate and solid material. Alternatively, thefermentation broth can be extracted directly.

It may be convenient to include an organic solvent extraction step inthe isolation or purification procedure, suitably using a solvent suchas acetone or hexane.

Further isolation of the desired compound may conveniently be effectedby chromatographic techniques. The extract may contain additionalsubstances, and therefore chromatographic separation may result in aplurality of fractions, of which the desired fraction or fractions is orare the fraction(s) comprising the desired compound or a derivativethereof.

The desired fraction(s) may readily be identified in a routine manner bytesting for anthelmintic activity and/or by monitoring each fractionchromatographically. The desired fraction(s) is/are that/thoseidentified by such procedures as containing the desired compound or aderivative thereof.

If necessary, repeated chromatographic separation may be carried out ina routine manner. At each stage of the separation procedure, thefractions containing the desired compound or a derivative thereof may becombined and then subjected to further purification steps. In theinitial separation steps, it may be convenient to identify the desiredfractions merely as those having anthelmintic activity and to combineall such fractions. In later stages of the separation, it may benecessary to identify the desired fraction or fractions more preciselyin order to separate the desired compound or a derivative thereof fromany other substances that may be present. Separation may advantageouslybe continued in order to give one or more fractions consistingessentially of the desired compound or a derivative thereof.

The expression `fraction consisting essentially of the desired compoundor a derivative thereof` means a fraction containing the desiredcompound or a derivative thereof as the sole component present in thatfraction, or as the major component (whether other components are activeor are inactive impurities) present in that fraction. The expression`major component` means the component that is present in the greatestamount relative to other individual components (exclusive of solvent).Suitably, the major component is present in an amount greater than thesum of the amounts of all other components (excluding solvent). Moresuitably, the major component is present in an amount of at least 60%,advantageously at least 70%, preferably at least 80%, especially from90% to 100%, by weight, relative to the total amount of active material,or relative to the total amount of material whether active or inactive(exclusive of solvent), as the case may be, present in the fraction.Typically, the compounds of the invention are produced in admixture withone another, so that fractions may be obtained which consist essentiallyof a mixture of two or more compounds of the invention.

It has been found convenient to carry out chromatographic separation onsilica gel (using, for example, a silica 60 column). Two or morechromatographic separation steps may be carried out successively.Elution of the chromatographic columns may conveniently be effectedusing organic solvents, either alone or in admixture with one another,e.g. hexane/acetone, diethylether/petroleum ether, ormethanol/chloroform.

The compound or mixture of compounds according to the invention issuitably provided in substantially pure form, for example at least 50%pure, suitably at least 60% pure, advantageously at least 75% pure,preferably at least 85% pure, more preferably at least 95% pure,especially at least 98% pure, all percentages being calculated asweight/weight. An impure or less pure form of a compound according tothe invention may, for example, be used in the preparation of a morepure form of the same compound or of a related compound (for example acorresponding derivative) suitable for pharmaceutical use.

The compounds of the invention have parasiticidal properties, forexample against nematodes such as Trichostrongylus colubriformis, andare useful for the treatment of helminthiasis in animals such as meals,including humans and domesticated animals (including farm animals).

Accordingly the present invention also provides a compound according tothe invention, for use in the treatment of the human or animal body,especially for treating endo- and ectoparasitic infestations andparticularly for treating helminthiasis of domestic and farm animals.

The term helminthiasis encompasses those diseases of man and animalscaused by infestation with parasitic worms such as Strongyles, Ascarids,hookworms lungworms, filarial worms and whipworms. The compound may alsobe used against nematodes occurring in the soil or parasitic to plants.

The compounds of the invention are also active against Arthropods. Thephylum Arthropoda comprises insects--such as biting flies, lice, bugs,beetles and fleas--and arachnids--such as mites and ticks.

Thus, a broad aspect of the invention provides a method of eradicatingarthropod or nematode infestations, which method comprises applying acompound according to the invention or a derivative thereof to thearthropods or nematodes or to their environment.

The present invention thus provides a pesticidal composition comprisinga compound according to the invention or a derivative thereof togetherwith a suitable carrier or excipient, such as an aerosol formulation.

The present invention also provides a pharmaceutical or veterinarycomposition comprising a compound according to the invention or apharmaceutically acceptable derivative thereof together with apharmaceutically or veterinarily acceptable carrier or excipient.

The present invention also provides a method of treatment or prophylaxisof endo- and ectoparasitic infestations, especially helminthiasis, ofanimals, especially humans and domesticated mammals, which comprisesadministering an effective non-toxic amount of a compound according tothe invention or a pharmaceutically acceptable derivative thereof, or acomposition according to the invention, to a patient in need thereof.

The composition according to the invention may be formulated foradministration in any convenient way for use in human or veterinarymedicine, by analogy with other anthelmintics.

In suitable formulations the drug may be administered to animals orally(as a paste, drench, bolus, capsule or tablet), parenterally,percutaneously, as a food additive (e.g. granules, pellets or powder),or may be prepared as an aerosol spray formulation.

The compounds of the invention may be formulated as a mixture with eachother and/or with other anthelmintics, insecticides, acaricides or otherpharmacologically active substances.

Suitably the composition consists of sufficient material to provide adose of from 0.01 to 100 mg of active ingredient per kg of animal bodyweight per dose, more suitably 0.1 to 10 mg/kg per dose.

A composition according to the invention may suitably contain from 0.1%by weight, preferably from 1.0 to 60% by weight, of the compoundaccording to the invention (based on the total weight of thecomposition), depending on the method of administration.

In certain circumstances the crude fermentation broth may beadministered, for example by incorporating the freeze-dried fermentationbroth into the feed of the animal.

It will be appreciated that, in some cases, it will be advisable torepeat the dosing of the infected or potentially infected human oranimal with the compound of the invention according to conventionaldosage regimes used with anthelmintics.

The following Examples illustrate the invention.

EXAMPLE 1 Production and Isolation of VM 44857

a) Fermentation

Streptomyces E225 was grown at 27° C. on a solid agar plate of starchcasein. A portion of growth taken from the agar plate was used toinoculate the seed medium (50 ml) contained in a 250 ml Erlenmeyerflask.

The seed stage was incubated at 27° C. on a gyratory shaker at 240r.p.m. for 48 hours. The seed medium had the following composition:

    ______________________________________                                        Constituent         Amount (g/l)                                              ______________________________________                                        Soybean flour       10.0                                                      Glycerol            20.0                                                      Maltose             2.0                                                       Trace element mix   10     ml stock/liter                                     Deionised water     to 1   liter                                              ______________________________________                                    

(The soybean flour was Arkasoy to as supplied by British Arkady Co.Ltd., Old Trafford, Manchester U.K.)

The trace element mix comprised:

    ______________________________________                                        Constituent    Amount (g/l)                                                   ______________________________________                                        CaCl.sub.2 2H.sub.2 O                                                                        10.0                                                           MgCl.sub.2 6H.sub.2 O                                                                        10.0                                                           NaCl           10.0                                                           FeCl.sub.3     3.0                                                            ZnCl.sub.2     0.5                                                            CuCl.sub.2 2H.sub.2 O                                                                        0.5                                                            MnSO.sub.4 4H.sub.2 O                                                                        0.5                                                            CoCl.sub.2 6H.sub.2 O                                                                        0.5                                                            ______________________________________                                    

The medium was adjusted to pH 6.5 before sterilisation.

Portions (2 ml) of the seed stage were used to inoculate thefermentation medium (50 ml) contained in 250 ml Erlenmeyer flasks. Thefermentation medium contained:

    ______________________________________                                        Constituent         Amount (g/l)                                              ______________________________________                                        Soluble starch      10                                                        Casein (Sigma C5890)                                                                              1.0                                                       Dipotassium hydrogen phosphate                                                                    0.5                                                       Magnesium sulphate  0.5                                                       ______________________________________                                    

(Sigma C5890 was supplied by Sigma Chemical Co. Ltd., Poole, Dorset,England "Sigma" is a Trade Mark).

The medium was adjusted to pH 7.0 before sterilisation.

The fermentation was incubated on a gyratory shaker at 240 r.p.m at 27°C. for 19 days.

Fermentation samples were assayed by testing for in vitro anthelminticactivity for example against Haemonchus contortus L₃ larvae.

After 19 days the whole broth from 100 fermentation flasks was combinedand centrifuged and the supernatant discarded.

b) Isolation of Substantially Pure VM 44857

The cell pellet obtained in (a) was slurried with water (0.75 l), mixedwith acetone (1.5 l) and allowed to stand for 48 hours at 4° C. Theslurry was then filtered (Hyflo supercel, BDH Chemicals Ltd., Eastleigh,Hampshire, England "Hyflo" is a Trade Mark), the residue washed withacetone (3×250 ml) and the combined filtrates were evaporated to removethe acetone.

The aqueous residue (600 ml) was mixed with methanol (500 ml) and thewhole extracted with hexane (3×500 ml). The combined hexane extractswere evaporated, the residue (412 mg) was dissolved in methanol (41.2ml) and allowed to stand at -20° C. overnight. After filtration toremove the precipitate which had formed, the filtrate was evaporated andthe residue (295 mg) chromatographed on silica (75 g) eluting with 0 to20% acetone in hexane. Fractions containing VM 44857, VM 44864 and VM44866, detected by t.l.c., were collected. The combined VM 44857fractions were evaporated to give the product (25 mg) as an oil. Finalpurification was effected by preparative thin layer chromatography usingsilica gel taper plates (ex Analtech, Anachem House, Luton,Bedfordshire, England) eluted with diethyl ether/petroleum ether 70/30to give VM 44857 (6 mg) R_(f) value in t.l.c. on a silica gel supportusing a diethyl ether/petroleum ether (70:30) solvent system=0.3.

Characterizing data

λ_(max) (CH₃ OH) 244 nm: m/z (FAB Na⁺ /Noba) (relative intensity) 591(100%) MNa!⁺ ; δ_(C) (CD₂ Cl₂) 173.2, 142.2, 139,5, 137.5, 136.6, 134.6,123.3, 123.0, 120.8, 119.7, 117.7, 97.4, 82.0, 79.8, 79.0, 68.4, 68.1,67.3, 67.3, 48.1, 45.4, 40.8, 36.1, 35.7, 35.3, 34.3, 31.2, 27.2, 21.8,19.2, 17.1, 14.9, 12.5, and 10.4 ppm. It has a retention time of 17.4minutes when subjected to hplc under the following conditions: anUltrasphere ODS 5 μm column (Altex 250×4.6 mm) is eluted withmethanol/water (90:10) at a flow rate of 1 ml/minute and monitored by UVabsorption at 246 nm. α!_(D) 25 (acetone)+111° (C=0.25).

EXAMPLE 2 Production and Isolation of VM 44864

The procedure described in Example 1 additionally yielded VM 44864 (4mg) R_(f) value in t.l.c. on a silica gel support using a diethylether/petroleum ether (80:20) solvent system=0.4.

Characterising data

λ_(max) (CH₃ OH) 244 nm and 237 nm; m/z (FAB Na⁺ /Noba) (relativeintensity) 621 (100%) MNa!⁺ ; α!_(D) ²⁵ (acetone)+96.8 (c=0.25); δ_(C)(CDCl₃) 173.8, 142.3, 139.8, 137.2, 135.9, 134.1, 123.6, 123.5, 120.5,119.5, 118.5, 98.6, 81.9, 80.3, 77.5, 76.9, 71.5, 68.6, 68.2, 68.0,57.7, 48.5, 45.6, 36.6, 36.4, 36.3, 35.9, 34.6, 32.1, 22.3, 19.9, 17.5,15.6, 13.1 and 10.9 ppm. It has a retention time of 7.0 minutes whensubjected to hplc under the conditions described in Example 1.

EXAMPLE 3 Production and Isolation of VM 44866

The procedure described in Example 1 additionally yielded VM 44866 (3mg) R_(f) value in t.l.c. on a silica gel support using a diethylether/petroleum ether (80:20) solvent system=0.25.

Characterizing data

λ_(max) (CH₃ OH) 244 nm and 236 nm; m/z (FAB Na⁺ /Noba) (relativeintensity) 607 (100%) MNa!⁺ ; α!_(D) ²⁵ (acetone)+81.9 (c=0.16); δ¹³ C(CDCl₃) 173.7, 142.7, 139.5, 137.8, 137.2, 134.1, 123.6, 123.4, 120.6,120.2, 118.0, 98.8, 81.9, 80.1, 79.1, 71.6, 68.6, 68.4, 68.0, 67.7,48.5, 45.7, 36.9, 36.5, 36.4, 36.0, 34.6, 32.1, 22.3, 19.9, 17.5, 15.5,13.1, 10.9 ppm. It has a retention time of 5.8 minutes when subjected tohplc under the conditions described in Example 1.

EXAMPLE 4 Production and Isolation of VM 44865

Portions (2 ml) of the seed stage obtained as described in Example 1were used to inoculate the fermentation medium (50 ml) contained in 250ml Erlenmeyer flasks. The fermentation medium contained:

    ______________________________________                                        Constituent           Amount (g/l)                                            ______________________________________                                        Casein hydrolysate    2.0                                                     Glucose               20.0                                                    Soluble starch        20.0                                                    Casein (Sigma C5890)  2.0                                                     Dipotassium hydrogen phosphate                                                                      0.5                                                     Magnesium sulphate    0.5                                                     Calcium carbonate     5.0                                                     Trace element mix as in Example 1                                                                   10     ml stock/liter                                   ______________________________________                                    

(Sigma C5890 was supplied by Sigma Chemical Co. Ltd., Poole, Dorset,England "Sigma" is a Trade Mark).

The medium was adjusted to pH 7.0 before sterilisation.

The fermentation was incubated on a gyratory shaker at 240 r.p.m at 27°C. for 13 days.

Fermentation samples were assayed by testing for in vitro anthelminticactivity for example against Haemonchus contortus L₃ larvae.

After 13 days the whole broth from 100 fermentation flasks was combinedand centrifuged and the supernatant discarded.

b) Isolation of Substantially Pure VM 44865

The cell pellet obtained in (a) was slurried with water (0.75 l), mixedwith acetone (1.0 l) stirred for 30 min and filtered. The acetoneextraction was repeated three times, and the combined filtratesevaporated to remove the acetone.

The aqueous residue (600 ml) was extracted with chloroform (4×500 ml).The combined chloroform extracts were dried (MgSO₄) and evaporated. Theresidue (6 g) was chromatographed on silica (100 g) and gradient elutedwith 0 to 100% ether in hexane. Fractions containing VM 44865, detectedby t.l.c., were combined and evaporated to give the crude product (520mg) as an oil. Final purification was effected by further columnchromatography using silica (75 g) eluted with 0 to 100% ether in hexaneto give VM 44865 (11 mg) R_(f) value in t.l.c. on a silica gel supportusing a dichloromethane/methanol (95:5) solvent system=0.4.

Characterizing data

λ_(max) (CH₃ OH) 244 nm; m/z (FAB Na⁺ /Noba) (relative intensity) 719(100%) MNa!⁺ ; δ¹³ C (CDCl₃) δ¹³ C 173.3, 167.5, 142.6, 139.4, 137.6,137.2, 134.9, 134.1, 128.4, 123.6, 123.4, 121.0, 120.5, 119.8, 98.8,81.9, 80.4, 77.3, 74.2, 71.5, 68.9, 68.3, 68.0, 64.3, 57.8, 48.5, 45.5,36.9, 36.4, 36.3, 35.9, 34.6, 32.1, 22.3, 17.5 15.6, 14.4, 13.1, 12.1,11.0 ppm. It has a retention time of 8.0 minutes when subjected to hplcunder the conditions described in Example 1.

EXAMPLE 5 Production and Isolation of VM 44867

(a) Fermentation

Streptomyces E225 was grown at 27° C. on an agar plate of starch caseinmedium. A portion of the culture was used to inoculate six 500 mlErlenmeyer flasks, each containing 100 ml of sterilised seed medium. Theflasks were incubated at 27° C. on a gyratory shaker at 240 rpm for 48hours.

The seed medium was made up as described in Example 1.

The flask contents were used to inoculate 151 seed medium in a 20 lstainless steel fermenter. The medium was of the same composition exceptthat tap water was used in place of deionised water. Prior tosterilisation the medium was pH 6.3 and was not adjusted. The seed stageculture was agitated at 200 rpm. and air was supplied at a rate of 15l/mm. Temperature was controlled at 28° C. Polypropylene glycol P2000(supplied by KWR Chemicals Ltd, Eleanor House, 33-35 Eleanor Cross,Waltham Cross, Herts.) was added automatically to control foaming.Incubation was continued for 48 hours at which time 4 l of the culturewas used to inoculate 100 l production medium contained in a 150 lstainless steel fermenter. The production medium had the followingcomposition:

    ______________________________________                                        Constituent             Amount (g/l)                                          ______________________________________                                        Potato Starch           20                                                    Casein                  2                                                     Glucose                 20                                                    Casein hydrolysate      2                                                     K.sub.2 HPO.sub.4       0.5                                                   MgSO.sub.4              0.5                                                   CaCO.sub.3              0.5                                                   Trace element mix as in Example 1                                                                     10     ml/l                                           Tap water               to 1   l                                              ______________________________________                                    

Prior to sterilisation the medium was pH 7.0

Potato starch and casein were supplied by British Drug Houses Ltd, BroomRoad, Parkstone, Poole, Dorset. U.K.

Casein hydrolysate was supplied by Oxoid Ltd, Wade Road, Basingstoke,Hampshire U.K.

The production stage culture was agitated at 160 rpm and air wassupplied at a rate of 50 l/min. Temperature was maintained at 28° C.Polypropylene glycol P2000 was added automatically to control foaming.The fermentation was harvested after 15 days and centrifuged to recoverthe mycelial mass.

(b) Isolation of VM44867

The mycelial mass obtained in (a) was extracted with acetone (2×40 l)and concentrated to remove the acetone. The residue (14 l) was thenextracted with dichloromethane (2×6 l), the combined extracts dried(MgSO₄) and evaporated to an oil (40 g).

The residue was chromatographed on silica and eluted sequentially with 0to 60% diethyl ether/hexane. Fractions containing VM 44867, as detectedby hplc, were combined and evaporated to give the semi-purified productas an oil (66 mg). Final purification was effected by preparative thinlayer chromatography using silica gel taper plates (ex Analtech, AnachemHouse, Luton, Bedfordshire, England) eluted withmethanol/dichloromethane 3:97 to give substantially pure VM 44867 (4.7mg) R_(f) value in t.l.c. on a silica gel support using amethanol/dichloromethane (3:97) solvent system=0.3.

Characterizing data

λ_(max) (CH₃ OH) 244 nm; M/_(Z) (FAB Na⁺ /Noba) (relative intensity) 623(100%) MNa!⁺. E.I. observed mass 600.3659 C₃₅ H₅₂ O₈ requires 600.3662.δ¹³ C (CDCl₃) 174.7, 140.4, 137.4, 136.1, 134.7, 134.0, 125.5, 124.7,123.7, 120.6, 118.8, 98.8, 81.8, 79.8, 77*, 71.6, 69.5, 68.1, 68.0,57.4, 48.6, 44.4, 36.9, 36.2, 36.2, 35.5, 34.5, 32.1, 21.4, 19.3, 17.5,16.1, 13.8, 13.1 and 10.9 ppm. It has a retention time of 9.5 minuteswhen subjected to hplc under the conditions described in Example 1.α!_(D) ²⁵ =+51° (acetone, c=0.21).

EXAMPLE 6 Production and Isolation of VM44868

(a) Fermentation

Streptomyces E225 was grown at 27° C. on an agar plate of starch caseinmedium. A portion of the culture was used to inoculate five 500 mlErlenmeyer flasks, each containing 100 ml of sterilised seed medium. Theflasks were incubated at 28° C. on a gyratory shaker at 240 rpm for 72hours.

The seed medium had the following composition:

    ______________________________________                                        Constituent           Amount (g/l)                                            ______________________________________                                        Oxoid Special Peptone*                                                                              2.5                                                     Oxoid Lab Lemco*      2.5                                                     Oxoid Tryptone*       2.5                                                     Oxoid Neutralised Soya Peptone*                                                                     2.5                                                     Soluble starch        2.5                                                     Glucose monohydrate   2.5                                                     Oxoid Malt Extract*   2.5                                                     Glycerol              2.5                                                     Trace Element Mix as in Example 1                                                                   10     ml stock/liter                                   Deionised water       to 1   liter                                            pH unadjusted.                                                                ______________________________________                                         *supplied by Oxoid Ltd, Wade Road, Basingstoke, Hampshire, UK.           

The flask contents were used to inoculate 151 seed medium in a 20 lstainless steel fermenter. The medium was of the same composition exceptthat tap water was used in place of deionised water. The seed stageculture was agitated at 200 rpm. and air was supplied at a rate of 15l/min. Temperature was controlled at 28° C. Polypropylene glycol P2000(supplied by KWR Chemicals Ltd, Eleanor House, 33-35 Eleanor Cross,Waltham Cross, Hefts.) was added automatically to control foaming.Incubation was continued for 48 hours at which time 4 l of the culturewas used to inoculate each of two 150 l stainless steel fermenterscontaining 100 l of the following production medium.

Fermentation medium contained:

    ______________________________________                                        Constituent           Amount (g/l)                                            ______________________________________                                        Dextrin.sup.(1)       25                                                      Black Treacle.sup.(2) 1.5                                                     CaCO.sub.3            1.25                                                    Glucose monohydrate   2.5                                                     Arkasoy `50`.sup.(3)  12.5                                                    P2000                 1.0                                                     Tap Water             to 1   liter                                            pH adjusted to 6.5                                                            ______________________________________                                         .sup.(1) 07005 Snowflake supplied by Corn Products (UK) Ltd Manchester.       .sup.(2) Supplied by Fowlers, Tate and Lyle Ltd., Croydon, Surrey, UK.        .sup.(3) Supplied by British Arkady Co. Ltd., Old Trafford, Manchester,       UK.                                                                      

One fermenter also contained 2.1% MOPS buffer together with 0.0125% K₂HPO₄. The other fermenter contained 0.05% K₂ HPO₄.

Sterile air was provided to each vessel at 50 liters per minute and apressure of 0.5 bar maintained in the head spaces. The cultures werestirred at 160 rpm initially, this was increased to prevent dissolvedoxygen levels falling below 20% of saturation. Fermentation temperaturewas 28° C.

The vessel containing MOPS buffer maintained a constant pH of 7.0±0.1.

The vessel without added MOPS buffer was fitted with automatic additionof hydrochloric acid (10%) and sodium hydroxide solution (10%) tocontrol pH in the range 6.8-7.2.

The fermentation broth was harvested from both fermenters after 168hours, together with the solids which had accumulated above the liquidlevel around the walls of each vessel. The broth was centrifuged torecover the mycelial mass.

(b) Isolation of VM44868

The mycelial mass obtained in (a) was extracted three times with acetone(751, 301, and 251) and the combined extracts concentrated to remove theacetone. The residue (271) was extracted twice with dichloromethane (161and 81), the extracts were combined and evaporated to give an oil (220g).

The residue was chromatographed on silica and eluted sequentially with 0to 100% diethyl ether in hexane. Fractions containing VM 44868, asdetected by hplc, were combined and evaporated to give the semi-purifiedproduct as an oil (900 mg). This was further purified by chromatographyon silica eluted sequentially with 0 to 1% methanol in dichloromethaneto give an oil (68 mg).

Final purification was effected by preparative thin layer chromatographyusing silica gel taper plates (ex Analtech, Anachem House, Luton,Bedfordshire, England) eluted with diethylether/hexane (4:1) to givesubstantially pure VM 44868 (26 mg) R_(f) value in t.l.c. on a silicagel support using a methanol/dichloromethane (7.5:92.5) solventsystem=0.45.

Characterising data

λ_(max) 237 nm(CH₃ OH); M/_(z) (FAB Na⁺ /NOBA) (Relative intensity) 591(100%) MNa!⁺, α!_(D) ²⁵ +108° (c,0.29 acetone), δ¹³ C (CDCl₃) 196.5,172.3, 144.8, 137.6, 136.8, 136.3, 136.0, 134.8, 130.4, 123.5, 123.0,121.0, 97.6, 82.4, 78.0, 69.5, 67.4, 58,0, 49.9, 48.3, 48.0, 41.0, 36.6,36.3, 35.6, 34.5, 31.5, 27.6, 21.7, 17.7, 16.0, 15.6, 13.0, 11.0, ppm.It has a retention time of 11.7 minutes when subjected to hplc under theconditions described in Example 1.

EXAMPLE 7 Biological Activity of VM 44857

Thirty 4-week-old Mongolian gerbils of mixed sex were each infected with750 Trichostrongylus colubriformis infective larvae (sheep strain) bygarage. Twenty days after infection these animals were randomlyallocated into six groups: two of four, three of five and one of seven.On the same day worm egg counts were carried out on pooled faecalsamples from each group to confirm that the infections had established.

At 21 days post-infection the animals were treated as follows:

Group 1: VM 44857 at 1 mg/kg

Group 2: VM 44857 at 0.25 mg/kg

Group 3: VM 44857 at 0.1 mg/kg

Group 4: VM 44857 at 0.025 mg/kg

Group 5: Albendazole at 0.25 mg/kg

Group 6: 50/50 mixture of DMSO/PEG 400 at 0.5 ml/gerbil

All treatments were administered orally by gavage.

VM 44857 was prepared as a 0.01% solution in a 50/50 mixture of DMSO andPEG 400. Dilutions of this solution were then prepared to provide theappropriate doses. The albendazole used was a dilution of proprietaryValbazen (Merck).

Three days after treatment worm egg counts were carried out on faecalsamples from each group to determine the effect of treatment. Allanimals were then necropsied for recovery and enumeration of worms. Thefollowing method was adopted.

Each small-intestine was removed separately and slit open longitudinallywith bowel scissors and jetted vigorously with water over a 150 microntest sieve. The washings retained on the sieve were concentrated andthen examined for worms in a glass dish under a low powerstereomicroscope. Assessment of activity was determined by comparison ofworm counts of treated and untreated animals. The results are summarisedin Table II.

                  TABLE II                                                        ______________________________________                                                     Number of             Activity                                   Treatment    Worms Recovered                                                                              Mean   %                                          ______________________________________                                        VM44857       0, 0, 1, 0, 0,                                                                              0.2    99.4                                       1 mg/kg                                                                       VM44857       0, 0, 0, 0,   0      100                                        0.25 mg/kg                                                                    VM44857       0, 0, 5, 0,   1.25   96.4                                       0.1 mg/kg                                                                     VM44857       4, 7, 8, 33, 9,                                                                             12.2   65                                         0.025 mg/kg                                                                   Albendazole  11, 1, 3, 9, 1,                                                                              5      85.6                                       0.25 mg/kg                                                                    Controls     39 56 45 13 35 17 38                                                                         34.7   --                                         DMSO/PEG400                                                                   ______________________________________                                    

EXAMPLE 8 Biological Activity of VM 44864

Fifteen 4-week-old Mongolian gerbils of mixed sex were each infectedwith 750 Trichostrongylus colubriformis infective larvae (sheep strain)by garage. Twenty days after infection these animals were randomlyallocated into three groups of five. On the same day worm egg countswere carried out on pooled faecal samples from each group to confirmthat the infections had established.

At 21 days post-infection the animals were treated as follows:

Group 1: VM 44864 at 1 mg/kg

Group 2: Ivermectin at 0.1 mg/kg

Group 3: 50/50 mixture of DMSO/PEG 400 at 0.5 ml/gerbil

All treatments were administered orally by gavage.

VM 44864 was prepared as a 0.01% solution in a 50/50 mixture of DMSO andPEG 400 and this solution was then diluted to provide the appropriatedose. The Ivermectin used was a dilution of proprietary Ivomec (Merck).

Three days after treatment worm egg counts were carried out on faecalsamples from each group to determine the effect of treatment. Allanimals were then necropsied for recovery and enumeration of worms asdescribed in Example 7. The results are summarised in Table III.

                  TABLE III                                                       ______________________________________                                                     Number                Activity                                   Treatment    of Worms Recovered                                                                           Mean   %                                          ______________________________________                                        VM44864      0, 1, 0, 0, 0, 0.2    98.0                                       1 mg/kg                                                                       Ivermectin   1, 1, 0, 2, 0, 0.8    91.0                                       0.1 mg/kg                                                                     Controls     3, 2, 5, 17, 18,                                                                             9.0    --                                         DMSO/PEG400                                                                   ______________________________________                                    

We claim:
 1. A compound of formula (I): ##STR10## wherein R¹ is hydrogenor methyl, R² is hydrogen or E 2-methyl 2-butenoyloxy, and R³ ishydrogen or hydroxy, with the proviso that when R³ is hydrogen, R¹ andR² are both hydrogen, and when R² is E 2-methyl 2-butenoyloxy, R¹ ismethyl; or a compound of formula (II): ##STR11## or a compound offormula (III): ##STR12##
 2. A compound according to claim 1, which isobtainable by the fermentation of Streptomyces E225 NCIB 12310 orStreptomyces E225B NCIB
 12509. 3. A pesticidal composition, comprising acompound according to claim 1 or a derivative thereof together with aninert carrier or excipient.
 4. A pharmaceutical or veterinarycomposition, comprising a compound according to claim 1 or apharmaceutically or veterinarily acceptable derivative thereof togetherwith a pharmaceutically or veterinarily acceptable carrier or excipient.